Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.
Bottom Line: Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface.Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side.We also determined that this constriction is located near the middle of the TH8 helix.
Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org.Show MeSH
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Mentions: We applied SCAM (Karlin and Akabas, 1998) to the TH6–TH7 segment in the hope of identifying channel-lining residues and perhaps also learning something about the secondary structure of this segment. We began by probing each mutant channel with the positively charged thiol-specific reagent MTS-ET. (MTS-ET had no effect on WT T-domain channels.) Fig. 2 shows a representative record of a macroscopic experiment, in this case with T-domain mutant L307C and trans MTS-ET. Note the characteristic His6-tag blocking at negative voltages and unblocking at positive voltage. We waited ∼10 min for the conductance at 30 mV to stabilize, and then added MTS-ET to the trans compartment. After a short delay because of the mixing time, the conductance dropped by a small amount, indicating reaction of trans MTS-ET with the cysteine residue. This is an indication that residue 307 is exposed in the channel. Table 1 summarizes the effects of cis and trans MTS-ET on the conductance of each mutant channel.
Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 email@example.com.